The Expression And Role Of SPARCL1 In Hilar Cholangiocarcinoma

ObjectiveHilar cholangiocarcinoma,also known as Klatskin tumor,refers to the carcinoma from common hepatic duct,the left or right hepatic duct and its confluence.Due to the late and unspecific symptoms,hilar cholangiocarcinoma is often in the advanced stage and has metastasized at the time of diagnosis with limited effect of treatment and poor prognosis.Therefore,it is clinically important to explore the mechanism of the occurrence and development of hilar cholangiocarcinoma,and find new biomarkers related to tumor recurrence and prognosis.Secreted protein acidic and rich in cysteines-like 1(SPARCL1)is an extracellular matrix glycoprotein,which is involved in the regulation of cell adhesion,migration and proliferation,and other biological functions.The expression of SPARCL1 can be detected in multiple organs.SPARCL1 has been thought to be a tumor suppressor according to the downregulation or absence of SPARCL1 expression in malignant tumors in colon,pancreas,stomach and other organs.But some researches have found that the expression of SPARCL1 in tumor cells is up-regulated.There are several studies disclosed that SPARCL1 are related to the tumor cell proliferation activity,process of vascularity and cellular cycle.However,the intensive study of SPARCL1 expression in hilar cholangiocarcinoma is limited so far.The role of SPARCL1 expression in the pathogenesis of hilar cholangiocarcinoma is unclear.Thus,this study will focus on the expression of SPARCL1 and its mechanism in hilar cholangiocarcinoma.MethodsIn our research,three main experiments were performed to study the expression of SPARCL1 in hilar cholangiocarcinoma and its mechanism in the tumor development.1.The expression of SPARCL1 was compared in 92 cases of hilar cholangiocarcinoma patients in general condition,tumor size,tumor stage(including T and N staging)and cell differentiation to analyze its correlation with the clinical pathological features.Immunohistochemical staining was used to detect the expression of SPARCL1 in 92 hilar cholangiocarcinoma patients,after which Western Blot was used to confirm it.2.SPARCL1 plasmid was transfected into QBC939 cells in vitro to study the effect of SPARCL1 overexpression in proliferation and metastasis of hilar cholangiocarcinoma.The expression of extracellular matrix metalloproteinase,including matrix metalloproteinase-9(MMP-9),matrix metalloproteinases-2(MMP-2),vimentin and fibronectin were detected to investigate the role of the changes of SPARCL1 expression on the occurrence and development of tumor cells.3.The correlation between the SPARCL1 expression in the hilar cholangiocarcinoma patients and the prognosis and survival was analyzed.Result 1.The expression of SPARCL1 was significantly correlated with lymph node metastasis and histological differentiation in hilar cholangiocarcinoma,while no significant correlation was found with gender,tumor size or tumor stage.Detected by immunohistochemical and confirmed by Western Blot analysis,SPARCL1 was strongly expressed in bile duct cells,while downregulated in hilar cholangiocarcinoma cell.Loss of SPARCL1 expression was observed in 50% of hilar cholangiocarcinoma.2.SPARCL1 plasmid transfected QBC939 cells had lower cell forming ability than the control group after 2 weeks in colony formation test.SPARCL1 plasmid transfected QBC939 cells had significantly less migrated cells than the control group in Transwell migration experiment.The result was further confirmed by wound healing assay.The overexpression of SPARCL1 can inhibit QBC939 cell migration.The tumor metastasis may be inhibited through the inhibition of MMP-9,MMP-2,vimentin and fibronectin.3.The hilar cholangiocarcinoma patients with positive expression of SPARCL1 had longer recurrence interval than the ones with negative expression of SPARCL1(32 months vs.12 months,p=0.001).The hilar cholangiocarcinoma patients with positive expression of SPARCL1 had longer median survival period than the ones with negative expression of SPARCL1(40 months vs.13 months,p<0.001).COX multivariate analysis showed that tumor invasion,lymph node metastasis and SPARCL1 expression were independent predictors of prognosis.Conclusion 1.The expression of SPARCL1 was downregulated in hilar cholangiocarcinoma patients.Moreover,absence of SPARCL1 was found in about half of the hilar cholangiocarcinoma patients.2.Overexpression of SPARCL1 can inhibit QBC939 cell’s proliferation and migration.The mechanism of inhibiting tumor metastasis may be explained by inhibition of MMP-9,MMP-2,vimentin and fibronectin.3.The hilar cholangiocarcinoma patients with positive expression of SPARCL1 have longer recurrence interval and longer survival than the negative ones.SPARCL1 can be a predictor of recurrence and survival in hilar cholangiocarcinoma.